Stabilized liquid egg material for extended shelf life

ABSTRACT

The present invention is directed to shelf-stable liquid egg material, methods for making the same, and method of using the same. The present invention is also directed to an animal feed supplement containing a stabilized IgY antibody titer in liquid eggs stored at room temperature for extended periods of time with the use of glycerol and preservatives. The stabilized liquid whole egg or stabilized liquid yolks of the egg containing non-specific or elevated specific IgY titer may be used as animal feed supplements animals to provide passive immunity to these animals. The stabilized nature of the IgY and liquid egg allows for extended shelf life of these liquid products at room temperature.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation under 35 U.S.C. §120 of U.S. patentapplication Ser. No. 12/980,536, filed Dec. 29, 2010, issued on Aug. 6,2013, as U.S. Pat. No. 8,501,220, which claims priority under 35 U.S.C.§119(e) to U.S. Provisional Patent Application 61/291,675, filed Dec.31, 2009, the entireties of all of which are incorporated herein byreference.

BIBLIOGRAPHY

Complete bibliographical citations to the documents cited herein can befound in the Bibliography immediately preceding the claims.

FIELD OF THE INVENTION

The present invention relates to shelf-stable liquid egg productscomprising IgY antibodies. The liquid egg products are formulated toretain IgY titer and activity while being stored at room temperature ina liquefied state. The present invention also relates to animal feedsupplements comprising the shelf-stable liquid egg products. Inparticular, the present invention relates to a stabilizing compositionfor adding to a whole egg or egg yolk. The composition componentscomplex with IgY to maintain IgY titer and activity for storage atextended periods of time.

BACKGROUND

IgY derived from hyperimmunized chicken eggs has been added as asupplement to animal feed to provide passive immunity in other animals.By a process of vaccination, chickens or other egg-laying animals can beimmunized against specific microorganisms and other antigens. Increasedtiters of antibodies can be obtained by a process of hyperimmunization.Hyperimmunization procedures are well-known in the art and have beendescribed in detail. High amounts of specific antibodies can be obtainedby immunizing animals with specific antigens and isolating theantibodies from the egg yolk, milk, colostrum or serum. While it hasbeen known since 1893 that the IgY antibody is found in the yolks ofchicken eggs (Klempeler 1893), it is only in the last 20 years or sothat use of IgY has gained ground as a practical source of passiveimmunity in livestock or other animals. While nature intended liquidcolostrum as the source of antibodies to provide the initial passiveimmunity that a newborn animal receives from its mother, in the modernagriculture industrial practice it has become necessary to providepassive immunity from various antibody supplements in order to achieveprofitability.

The supplements that the animal feed industry uses to provide passiveimmunity to animals have been derived from colostrum, blood serum and,more recently, from chicken egg IgY antibodies. The use of IgYantibodies to provide passive immunity provides certain advantages overthe use of antibodies found in colostrum or antibodies found in bloodserum. These advantages include the relative cost efficiency to createspecifically directed antibodies against pathogenic organisms comparedto colostrum or blood serum antibodies.

Passive immunity is the transfer of active humoral immunity in the formof ready-made antibodies, from one individual to another. Passiveimmunity can occur naturally, when maternal antibodies are transferredto the offspring. It can also be induced artificially, when high levelsof antibodies specific for a pathogen or a toxin are recovered fromimmunized individuals and administered to non-immune individuals. Theantibodies' transfer may be carried out via systemic, intravenous ororal routes. The oral route is the route of choice for localizedtreatment of digestive tract infections. Immunity derived from passiveimmunization lasts for only a short period of time, i.e., for as long asthe antibodies remain in the organism, but it provides immediateprotection.

In animals, the administration of preformed, specific antibodies is anattractive approach to establish protective immunity against viral andbacterial pathogens. It is becoming a more interesting alternative tocontrol the increasing number of antibiotic-resistant organisms. Passiveimmunization can also be used against organisms that are non-responsiveto antibiotic therapy.

Antigen-specific IgY antibody can be produced on a large-scale from eggslaid by chickens immunized with selected antigens (Hatta et al., 1997).The laying hen transfers all antibody isotypes found in the chicken tothe egg, i.e., IgY, IgM, and IgA antibodies. The yolk contains only IgY,while IgM and IgA are found only in the white. The chicken's serum IgYlevel is reflected in the egg yolk shortly after a single administrationof antibody, about one week. Egg yolk contains approximately 3-25 mgIgY/ml. Depending on its weight, therefore, each egg could provide40-500 mg IgY.

The use of IgY for passive immunization has been studied extensively,demonstrating its effectiveness in preventing or treating infectiousdiseases caused by various pathogens in animal models, especially thoseof the gastrointestinal (GI) tract. Antibodies are usually administeredin the feed in several forms: whole egg powder, whole yolk powder,water-soluble fraction powder or purified IgY material. The powdered eggformats containing IgY or other anti-bacterial components are typicallypackaged into containers or made into pastes. The powders are mixed intodry feed and subsequently given to the animal. The pastes are squirtedinto the mouth of the animal.

The animal feed industry also uses liquid feed supplements as a standardmethod of providing supplements to animals, due to cost considerationsand ease of use considerations. A liquid format is desirable since it iseasy to administer liquids in drenches or even in watering systems.However, a stabilized liquid format that maintains titers of IgY derivedfrom hyperimmunized chicken eggs for an extended shelf life has not beendeveloped.

The methods that are currently used to preserve IgY titer from eggsinvolve either expensive spray-drying or freeze-drying techniques forthe whole egg or egg yolk and converting the egg into a powder. However,both of these methods have problems in maintaining the originalstructure of the IgY and binding activity of the IgY molecule.Spray-drying methods use heat during the processing of the egg materialthat will denature the IgY molecule to a large extent. IgY denatureswhen thermally treated at temperatures higher than 75° C. (Chang et al.,1999). Freeze-drying methods involve freezing the egg material whichalso can structurally alter the IgY molecule. Chansarkar (1998) showedthat frozen or freeze-dried IgY resulted in loss of antigen-bindingactivity and a significant drop in the solubility under the conditionsof high salt and protein concentrations.

Keeping the IgY antibody in a liquid state from the time it is in theegg to the time it is used is the best method to maintain the structureof the IgY molecule, to prevent degradation, and preserve its bindingactivity in providing passive immunity. Keeping the hyperimmune eggs intheir egg shell without cracking them open is not a practical commercialmethod. Whole eggs stored at room temperature will naturally startdegrading within several weeks and are also not practical to use as adelivery means to the animal.

Keeping whole egg or yolk products in a stable liquid state at roomtemperature without degradation of the liquid matrix or the IgY contentfor periods of time greater than few hours or even a day is a problem tothe animal feed industry. This is due to oxidation issues, contaminationissues, and coagulation issues of liquid egg products. Attempts atkeeping whole eggs or liquid yolks as a stabilized liquid matrix havebeen the subject of a number of patents for human products. Thesemethods principally involve pasteurization of the egg product at varioustemperatures. For example, U.S. Pat. No. 6,403,141 is directed to amethod of obtaining a long shelf life for liquid egg products usingpasteurization techniques. This method allows storage of whole eggproducts at room temperature for 3 months without the incorporation ofadditives. However, this patent has not addressed the overriding issueof IgY stability when storing the products at room temperature and use adifferent technology approach than the current invention to extendingshelf life of the matrix.

Other methods of purifying IgY on a commercial scale may lose importantfactors that may be involved in the efficacy of whole chicken eggs orwhole yolks when providing antibacterial properties. While IgY is thefocus of passive immunity, it has also has been shown thatanti-bacterial properties were associated with egg yolk components otherthan the IgY. Among these components are the granule high-densitylipoprotein (HDL) (Kassaify et al., 2005), the plasma low-densitylipoprotein (LDL) (Brady et al., 2002), and the egg-yolk-derivedsialyloligosaccharide (YDS) (Sugita-Konish et al., 2002). As such, itdesirable to maintain these components when providing passive immunityin a liquid egg product by making sure the entire yolk is used or theentire contents of the egg are used and not using just purified IgY.

SUMMARY OF THE INVENTION

It is an object of this invention to increase the shelf life of one ormore antibodies, including IgY, obtained from the egg of anegg-producing animal that has been immunized against one or moreimmunogens, when kept in a liquid state. In particular, the invention isdirected to increasing the stability and maintaining activity of the IgYin a liquid egg material.

In one broad aspect, the present invention is directed to a shelf-stableliquid egg material. The shelf-stable liquid egg material comprises aliquid egg material enriched with an antibody at a hyperimmunized titer,in addition to additional ingredients selected from the group consistingof an antibody protectant, an antioxidant, an antimicrobial agent, anenzyme inhibitor, and an alkalinizing agent. The additional ingredientsare present in concentrations effective to extend shelf life of theliquid egg material and/or the antibody contained therein.

In another broad aspect, the invention is directed to a method of makinga shelf-stable liquid egg material as described herein. The methodcomprises inducing an immune response in an egg-producing animal with atleast one antigen to produce an antibody at a hyperimmunized titer;collecting an egg from the egg-producing animal following induction ofthe immune response; processing inner content of the egg to obtain aliquid egg material; and adding to the liquid egg material an antibodyprotectant, an antioxidant, an antimicrobial agent, an enzyme inhibitor,and an alkalinizing agent in concentrations effective to extend shelflife of the liquid egg material.

In yet another broad aspect, the invention is directed to a method ofusing a shelf-stable liquid egg material as described herein. The methodcomprises administering the liquid egg material to an animal to providepassive immunity to the animal.

The present invention provides a method of maintaining the IgY titer andmaintaining the liquid egg matrix at room temperature for an extendedshelf life without having to use expensive and sometimes detrimentalspray-drying or freeze-drying techniques. These advantages are importantwhen commercializing an IgY product as a liquid animal feed supplement.

Advantageously, the processed liquid egg material of the presentinvention may be stored in a stabilized blend at room temperature for atleast 6 months to a year, 1 to 2 years when kept refrigerated, andgreater than 2 years when kept frozen, and still maintain the bindingactivity of the antibody and matrix stability of the liquid eggmaterial.

The objects and advantages of the invention will appear more fully fromthe following detailed description of the preferred embodiment of theinvention.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless otherwise noted, the following definitions will apply to theterms throughout this disclosure:

“Antimicrobial preservatives and agents” mean agents that kill microbes,i.e., biocidal agents, and agents that retard microbe growth, i.e.,static agents. As used here, “microbes” means bacteria, fungi, viruses,prions, protozoa or any other microscopic organism capable of spoilingfood.

“Content” or “contents” of an egg refers to any portion of the egg, inwhole or in part. In a preferred version of the invention, the contentof an egg comprises the inner content of the egg. “Inner content” of anegg refers to any portion of the egg, in whole or in part, minus theshell, and includes the egg whites and/or the egg yolks.

“Egg-producing animal” refers to any egg-producing or egg-laying animalcapable of producing antibodies to an antigen and passing along theantibodies in its eggs. Avian egg-producing animals are preferred.Non-limiting examples of suitable avian egg-producing animals includechickens, turkeys, ducks, and ostriches.

“Processing” inner content of an egg refers to any method of obtainingand blending the inner content of the egg into a relatively homogeneous,liquid form. Such processing includes, without limitation, cracking theegg to obtain the inner content, and blending the inner content usingmethods known in the art. If inner content from more than one egg isextracted, then the total collected inner content may be processed toform a blend of liquid egg material.

“Hyperimmunization” is the process of repeated injections of an antigenleading to high levels of an antibody in a body.

“Hyperimmunized titer” refers to a level of an antibody against anantigen in an animal as would result from hyperimmunization of theanimal with the antigen.

“IgY and other antibodies” refers to the classes of immunoglobulinsfound in chickens or other avian egg-producing animals and comprisesIgY, IgA and IgM.

“Induction of an immune response” refers to any process of stimulatingproduction of antibodies, preferably against a specific antigen orantigens, in an animal. Methods include any known or developed methodsof vaccination or immunization. In a preferred version, induction of animmune response comprises injecting the particular antigen or antigensin an animal, which causes the animal to produce an antibody against theantigen or antigens. “Immunogen is used herein interchangeably with“antigen.”

“Liquid egg material” refers to inner content of an egg that has beenextracted from the egg and, if necessary, processed into liquid form.Liquid egg material may include the yolk, egg whites, and/or any otheradditives or ingredients described herein.

“Liquid whole egg” means a liquid egg material comprising liquid eggwhite and liquid egg yolk.

“Liquid egg yolk” means liquid egg yolk that is substantially free ofliquid egg white. Liquid egg yolk can be obtained by separating the yolkfrom egg white and processing the egg yolk into liquid form.

“Liquid egg white” means liquid egg white substantially free of egg yolkor liquid egg yolk. Liquid egg white may be obtained by separating theegg white from the egg yolk. Processing of the egg white may berequired.

Described in this disclosure is a method for extending the shelf life ofliquid egg material, without the need of heat treatment. Theshelf-stable liquid egg material comprises the inner content of eggs(whole egg, egg yolk, and/or egg whites) in combination with additionalingredients such as protectants, antioxidants, and preservatives. Theadditional ingredients are added to stabilize the IgY and otherantibodies within the liquid egg material to ensure an extended shelflife when stored at room temperature, refrigerated or frozen. Thestabilized IgY in the egg is then used to provide passive immunity inanother animal. At least some of the ingredients additionally enhancethe efficacy of the liquid egg material in the animal.

It is well known that antibody produced in one species can be used toneutralize the effects of the corresponding antigen in other species.Passive immunization occurs when an individual receives immuneprotection from antibodies produced in another individual. Theseindividuals can be of the same species or from different species. Forexample, chicken hens may be immunized with a vaccine for calf scours inorder to obtain high amounts of specific antibodies against pathogenicE. coli strains that cause scours in calves. The antibody-containingyolk obtained from an egg of the immunized hen will have activityagainst such E. coli strains. If administered to calves, theantibody-containing yolk is effective in protecting calves from attackby the scours-causing bacteria.

One specific version of the present invention is directed to a methodfor increasing the stability of the IgY in a liquid egg material to forma stabilized blend of liquid egg material and administering thestabilized blend of liquid egg material to an animal, comprising:

A. inducing an immune response in an egg-producing animal with at leastone immunogen;

B. collecting eggs from the egg-producing animal for a predeterminedperiod of time following induction of the immune response;

C. processing all of the collected eggs together to form a blend ofliquid whole egg material or liquid yolk material;

D. adding a protectant to stabilize and maintain the structuralintegrity of the antibody and the integrity of the other contents of theblend;

E. adding antioxidants to the blend;

F. adding antimicrobial preservatives to the blend;

G. adding digestive enzyme inhibitors to the blend;

H. adding alkalinizing agents to the blend;

I. storing the stabilized blend in suitable containers for an extendedshelf life; and

J. administering to the stabilized blend to an animal to provide passiveimmunity.

Liquid Egg Material:

The present invention includes a liquid egg material prepared from wholeeggs, yolks of eggs, and/or egg whites. The liquid egg materialpreferably contains IgY and/or other antibodies, preferably at least IgYantibodies, from hyperimmunized chickens or other egg-producing animals.The liquid egg material may include content derived from whole eggs, eggyolks, or egg whites, provided that the liquid egg material contains IgYor other antibodies from the immunized animals. Preferably, the liquidegg material includes content derived from egg yolks.

Eggs are collected from the egg-producing animal after immunization. Theeggs are preferably collected for a predetermined period of time afterimmunization wherein the IgY titers in the eggs are the highest. Thispredetermined period of time can be determined by testing the IgY tiersby ELISA or other methods known in the art. The resultant IgYantibody-containing eggs are cracked open to obtain the entire liquidcontent inside them. Preferably, the whole-egg liquid content isadditionally separated to obtain just the yolk, where IgY antibodies arepredominantly found.

Antibody Protectants

The IgY is stabilized by adding antibody protectants. Non-limitingexamples of such antibody protectants include glycerol, propyleneglycol, ethylene glycol, or combinations thereof. These protectantsserve to maintain the antibody structure. The protectants also serve tomaintain the liquid egg material matrix and thus act as stabilizers.Along with water, the protectants act to promote softness andflexibility of the liquid egg material. This prevents drying out ofproteins such as IgY or other compounds found in the egg matrix.Preservation of the IgY structure and activity is maintained bypreventing such drying.

The antibody protectants can be added to the liquid egg product in anyeffective concentration to extend the shelf life of the liquid eggproduct. For example, the range of protectants added to the liquid eggmaterial can be from about 1% to 60% v/v, preferably from about 10% to40% v/v, and more preferably from about 15% to about 30% v/v.

The preferred antibody protectant is glycerol in an amount of about 20%v/v.

Antimicrobial Agents:

Antimicrobial agents are added to the liquid egg material to prevent thegrowth of bacteria, viruses, molds or fungus during long term storage.The antimicrobial agents added to the liquid egg material are preferablyfood grade material, i.e., capable of being ingested by an animalwithout causing deleterious health effects.

Non-limiting examples of antimicrobial agents include sugars such assucrose or salts such as sodium chloride, which work through osmoticmeans to destroy bacteria. Other antimicrobial agents such as potassiumsorbate, methylparaben or propylparaben can inhibit the growth of fungusand molds. Natural anti-bacterial agents, such as essential oils, can beadded to prevent bacterial contamination. Examples of suitable essentialoils include oregano, garlic, thyme, rosewood, celery seed,frankincense, yiang yiang, cedarwood, lime, orange, petitgrain,bergamot, lemon, grapefruit, mandarin, myrrh, coriander, pumpkin,cypress, lemongrass, palmarosa, citronella, carrot seed, eucalyptus,fennel, wintergreen, juniper, French lavender, Tasmanian lavender,macadamia, tea tree, cajuput, niaouli, peppermint, spearmint, basil,evening primrose, marjoram, geranium, aniseed, bay, pine, black pepper,patchouli, apricot kernel, sweet almond, rosemary, sage, clary sage,sandalwood, clove, vetiver, and ginger oils.

The antimicrobial agents can be added to the liquid egg product in anyeffective concentration to extend the shelf life of the liquid eggproduct.

For example, suitable concentrations of sucrose range from about 1% w/vto 60% w/v, preferably from about 5% w/v to about 30% w/v and mostpreferably from about 10% w/v to about 15% w/v.

Suitable concentrations of sodium chloride range from about 1% w/v to30% w/v, preferably from about 5% w/v to about 20% w/v and mostpreferably about 10% w/v.

Suitable concentrations of potassium sorbate range from about 0.01% w/vto 0.1% w/v, and preferably about 0.1% w/v.

Suitable concentrations of propylparaben and methylparaben range fromabout 0.01% w/v to 0.1% w/v, preferably from about 0.05% w/v to about0.1% w/v and most preferably from about 0.08% w/v to about 0.1% w/v.

Suitable concentrations of essential oils range from about 0.0001% v/vto 5% v/v, preferably from about 0.1% v/v to about 2% v/v and mostpreferably from about 0.5% v/v to about 1% v/v.

Antioxidants:

Antioxidants are added to the liquid egg material to prevent oxidationand degradation of IgY, other antibodies, and other components of theliquid egg material. Examples of suitable antioxidants includetocopherol, gallic acid ester, dibutyl hydroxy toluene (BHT), butylhydroxy anisol (BHA), ascorbic acid (vitamin C), vitamin E, andcombinations thereof.

The antioxidants can be added to the liquid egg product in any effectiveconcentration to extend the shelf life of the liquid egg product.

Suitable concentrations of vitamin E and vitamin C range from about0.01% w/v to about 5% w/v, and preferably about 0.1% w/v.

Suitable concentrations of dibutyl hydroxyl toluene (BHT) and butylhydroxyl anisol (BHA) range from about 0.01% w/v to about 0.02% w/v,preferably from about 0.015% w/v to about 0.018% w/v and most preferablyfrom about 0.016% w/v to about 0.017% w/v.

Digestive Enzyme Inhibitors:

The preferred method of administering the liquid egg material of thepresent invention to an animal is through oral administration byincluding it as a feed supplement. For the IgY feed supplement to beoptimally effective, the IgY should survive passage through the stomachand reach the gastrointestinal tract. The stomach of an animal containsenzymes and acids that help digest food, which would potentially damagethe IgYs in the administered liquid egg material.

Digestive enzyme inhibitors are added to the liquid egg material toprevent digestion of the IgY in the stomach of the animal in which theliquid egg material is orally administered. Examples of suitabledigestive enzyme inhibitors include soybean trypsin inhibitor, achymotrypsin inhibitor, a carboxypeptidase inhibitor, a papaininhibitor, a pepsin inhibitor, and other protease or peptidaseinhibitors. Preferably, the digestive enzyme inhibitor is soybeantrypsin inhibitor. Trypsin inhibitors inhibit the absorption of proteinsby reducing the activity of the enzyme trypsin, which is necessary fordegrading proteins to amino acids. This allows the IgY antibody to passthrough the stomach of the animal and into the gastrointestinal tract.Once the IgYs are in the gastrointestinal tract, they can interact withspecific pathogens in the gastrointestinal tract, such as E. coli, andprovide passive immunity to the animal.

Additionally, the digestive enzyme inhibitors help protect againstnative protease or peptidase enzymes in the liquid egg material.

The digestive enzyme inhibitors can be added to the liquid egg productin any effective concentration to extend the shelf life of the liquidegg product and/or to provide passive immunity to an animal to which theliquid egg material is administered.

Suitable concentrations of the digestive enzyme inhibitors range fromabout 0.0001% w/v to about 0.1% w/v, preferably from about 0.001% w/v toabout 0.05% w/v and most preferably from about 0.03% w/v to about 0.04%w/v.

Alkalinizing Agents:

Alkalinizing agents are added to the liquid egg material to preventacid-dependent denaturization of IgYs in the stomach of an animal towhich the liquid egg material is administered. Alkalinizing agents caninclude sodium carbonate, sodium bicarbonate, calcium carbonate,magnesium carbonate or combinations thereof. Alkalinizing agents raisethe pH of the stomach by interacting with stomach acids and neutralizingthem. This allows the IgY antibodies to be protected when passingthrough the animal stomach to the gastrointestinal tract to providepassive immunity to the animal. The alkalinizing agents also maintainthe pH of the liquid egg material while in storage to extend its shelflife.

The alkalinizing agents can be added to the liquid egg product in anyeffective concentration to extend the shelf life of the liquid eggproduct and/or to provide passive immunity to an animal to which theliquid egg material is administered.

Suitable concentrations of alkalinizing agents range from about 2.0% w/vto about 10% w/v, preferably from about 3% w/v to about 8% w/v and mostpreferably from about 4% w/v to about 6% w/v.

Preferred Concentrations:

A preferred formula for the liquid egg material includes the followingrecipe:

-   -   10% w/v sodium chloride;    -   20% v/v glycerol;    -   0.1% w/v potassium sorbate;    -   0.1% w/v vitamin E;    -   0.1% w/v vitamin C;    -   0.1% propylparaben; and    -   0.1% methylparaben.

Storage:

Once the protectant, stabilizers, antimicrobials, antioxidants, andalkalinizing agents are added and mixed into the liquid egg material,the liquid egg material is packaged in suitable storage containers.

The containers can be stored frozen, refrigerated or at roomtemperature. Storing the shelf-stable liquid egg material at roomtemperature is an advantage as storage costs are considerably lesswithout the need for refrigeration. In addition, the ability to storethe liquid egg material at room temperature renders it useful for farmsthat do not have refrigeration readily available, as found in developingcountries. While the liquid egg material of the present invention can bestored for extended periods at room temperature, refrigeration orfreezing of the liquid egg material can extend the shelf life evenfurther.

Preferred Recipe of Liquid Egg Material and Method of Making Same:

Whole eggs are cracked open manually or using automated methods into acontainer at room temperature. The yolks can be separated into aseparate container and processed as a yolk-only material, if desired.

Glycerol, sodium chloride, potassium sorbate, vitamin E, vitamin C,propylparaben, and methylparaben are added at room temperature to theliquid egg material and mixed. These ingredients can be added in anyorder as long as thorough mixing is achieved. In a preferred version,glycerol is added first, followed by sodium chloride, potassium sorbate,vitamin E, vitamin C, propylparaben, and methylparaben to ensurethorough dissolution of each ingredient. Thorough mixing is carried outto dissolve solid ingredients using a blender or other automated mixingequipment known to the art. The mixed liquid egg material is packagedinto light-proof containers and stored at a desired storage temperature.

Use of Invention:

The liquid egg material of the present invention can be used to providepassive immunity to any animal and is particularly useful withlivestock. The liquid egg material of the present invention is suitablefor use on all mammals including but not limited to the bovine species,ovine species, canine species, feline species, caprine species, swinespecies and equine species, all avian species including chickens andturkeys, all reptile species, all fish species, and all invertebratespecies. The preferred methods of administering the liquid egg materialto the animal includes oral administration, although other methods suchas parenteral administration are also acceptable.

Use of the liquid egg material of the present invention to providepassive immunity to livestock is achieved by pouring the invention intowatering systems or by directly drenching animals. The liquid eggmaterial may be given directly to the animal; mixed into dry feed; mixedinto liquid feed; or mixed into water and then given to the animal.Non-limiting examples of preferred administrations include thefollowing:

For poultry, 8 ounces of the liquid egg material is mixed with 1 gallonof water to form a stock solution. The stock solution can then bemetered into drinking water at the rate of 1 ounce of stock solution pergallon of drinking water.

For swine, a stock solution can be prepared and metered at the rate of 1gallon stock solution per 128 gallons of water through a medicator. Aproposed stock solution formula is prepared according to the followingrecommendations:

Sows and Boars: 8 ounces of the liquid egg material to 1 gallon ofwater;

Nursery: 16 ounces of the liquid egg material to 1 gallon of water;

Growers and Finishers: 8 ounces of the liquid egg material to 1 gallonof water.

For the bovine species, it is preferred to add 4 ml of the liquid eggmaterial of the present invention to milk or milk replacer once per calfdaily for 7 days, followed by 2 ml per day until weaning. This isaccomplished by making a stock solution of formula of the presentinvention and metering to calves at the rate of 1 gallon of stocksolution per 128 gallons of milk or milk replacer once daily. Apreferred concentration follows. For the first 7 days after birth, wateris added to 32 ounces of the liquid egg material of the invention untilthe volume makes up 1 gallon of stock solution. From day 8 untilweaning, water is added to 16 ounces of the liquid egg material of theinvention until the volume makes up 1 gallon of the stock solution.

The liquid egg material of the present invention provides considerableease of use to the end user to provide specific IgY passive immunity.

Unless explicitly indicated otherwise, all concentrations describedherein indicate the final concentration in the liquid egg material.References to percent weight per volume (w/v) or volume per volume (v/v)indicates weight or volume, respectively, of the added component perfinal volume of liquid egg material.

The ingredients and method steps described herein can be used in anycombination whether explicitly described or not.

All combinations of method steps as used herein can be performed in anyorder, unless otherwise specified or clearly implied to the contrary bythe context in which the referenced combination is made.

As used herein, the singular forms “a,” “an,” and “the” include pluralreferents unless the content clearly dictates otherwise.

Numerical ranges as used herein are intended to include every number andsubset of numbers contained within that range, whether specificallydisclosed or not. Further, these numerical ranges should be construed asproviding support for a claim directed to any number or subset ofnumbers in that range. For example, a disclosure of from 1 to 10 shouldbe construed as supporting a range of from 2 to 8, from 3 to 7, from 5to 6, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.

All patents, patent publications, and peer-reviewed publications (i.e.,“references”) cited herein are expressly incorporated by reference tothe same extent as if each individual reference were specifically andindividually indicated as being incorporated by reference. In case ofconflict between the present disclosure and the incorporated references,the present disclosure controls.

The methods, compounds, and compositions of the present invention cancomprise, consist of, or consist essentially of the steps, ingredients,and limitations described herein, as well as any additional or optionalsteps, ingredients, or limitations described herein or otherwise usefulin the art.

EXAMPLES

The following Examples demonstrate how the present invention has beenpracticed, but should not be construed as limiting.

Example 1

Egg-laying chickens were hyperimmunized with a commercially availablecoronavirus vaccine. After a suitable time to allow specific IgY titerto coronavirus to increase, the eggs were collected and cracked open toobtain the entire liquid egg contents of the whites and yolk. Theresultant volume of the whole egg-derived liquid egg material wasmeasured. Glycerol was added and mixed into the liquid egg material to afinal concentration of 20% v/v. Sodium chloride was then mixed into theliquid egg material to a final concentration of 10% w/v, along withpotassium sorbate at 0.1% w/v, vitamin C at 0.1% w/v, vitamin E at 0.1%w/v, sodium carbonate at 2% w/v, and soybean trypsin inhibitor at 0.1%w/v. This liquid egg material was then subjected to analysis for IgY tospecific coronavirus by Enzyme Linked Immuno Assay (ELISA) to generate abaseline IgY titer concentration.

Aliquots of the liquid egg material were stored frozen at −20° C.,refrigerated at 4° C., or at room temperature at 22° C., 37° C. or 55°C. Periodic sampling occurred weekly over the course of 6 weeks. and thesamples were analyzed by ELISA for IgY titer concentrations. No changeswere observed in the IgY titer concentrations. Accelerated stabilitystudies using Arrhenius plot calculations indicate the stability of thespecific IgY for coronavirus is greater than 1 year at room temperatureunder the conditions used. The matrix of the whole egg-derived eggmaterial remained liquid, and no microbial growth occurred at anytemperature used. The frozen storage conditions showed no changes in IgYtiter upon thawing, indicating stability of the IgY in the liquid eggmaterial of the present invention even when frozen and then thawed.

Example 2

Egg-laying chickens were hyperimmunized with a commercially availablecoronavirus vaccine. After a suitable time to allow specific IgY titerto coronavirus to increase, the eggs were collected and cracked open toobtain just the yolk. The resultant volume of the yolk-derived liquidegg material was measured. Glycerol was added and mixed into the liquidegg material to a final concentration of 20% v/v. Sodium chloride wasthen mixed into the liquid egg material to a final concentration of 10%w/v, along with potassium sorbate at 0.1% w/v, vitamin C at 0.1% w/v,vitamin E at 0.1% w/v, sodium carbonate at 2% w/v, and soybean trypsininhibitor at 0.1% w/v. The liquid egg material was then subjected toanalysis for IgY to specific coronavirus by Enzyme Linked Immuno Assay(ELISA) to generate a baseline IgY titer concentration.

Aliquots of this final liquid matrix were stored frozen at −20° C.,refrigerated at 4° C., or at room temperature at 22° C., 37° C., or 55°C. Periodic sampling occurred weekly over the course of 6 weeks, and thesamples were analyzed by ELISA for IgY titer concentrations. No changeswere observed in the IgY titer concentrations. Accelerated stabilitystudies using Arrhenius plot calculations indicate the stability of thespecific IgY for coronavirus is greater than 1 year under the conditionsused. The matrix of the yolk-derived egg material remained liquid, andno microbial growth occurred at all temperatures. The frozen storageconditions showed no changes in IgY titer upon thawing, indicatingstability of the IgY in the liquid egg material of the present inventioneven when frozen and then thawed.

The present invention is not intended to be limited to the foregoingexamples, but to encompass all such modifications and variations as comewithin the scope of the appended claims. It is understood that theinvention is not confined to the particular construction and arrangementof parts herein illustrated and described, but embraces such modifiedforms thereof as come within the scope of the claims following the citedreferences.

BIBLIOGRAPHY

-   Chang H. M., Ou-Yang R. F., Chen Y. T. & Chen C. C., 1999.    Productivity and some properties of immunoglobulin specific against    Streptococcus mutans serotype C in chicken egg yolk (IgY). J. Agric.    Food Chem., 47, 61-66.-   Chansarkar N. L., 1998. Studies on structural stability of hen's egg    yolk immunoglobulin (IgY). PhD Thesis: University of British    Columbia (Canada).-   Hatta H. et al., 1997. Passive immunization against dental plaque    formation in humans: effect of mouth rinse containing egg yolk    antibodies (IgY) specific to Streptococcus mutans. Caries Res., 31,    268-274.-   Kassaify Z. G., Li E.& Mine Y., 2005. Identification of    anti-adhesive fraction(s) in non-immunized egg yolk powder: in vitro    study. J. Agric. Food Chem., 53, 4607-4614.-   Klemperer F., 1893. Ueber natürliche Immunitüt and ihre Verwertung    für die Immunisierungstherapie. Arch. Exp. Pathol. Pharmakol., 31,    356-382.-   J. D. Quigley, K. R. Martin, H. H. Dowlen, and K. C. Lamar. 1995.    Addition of Soybean Trypsin Inhibitor to Bovine Colostrum: Effects    on Serum Immunoglobulin Concentrations In Jersey Calves. J Dairy Sci    78:886-892.-   Sugita-Konishi Y. et al., 2002. Inhibition of bacterial adhesion and    Salmonella infection in BALB/c mice by sialyoligosaccharides and    their derivatives from chicken egg yolk. J. Agric. Food Chem., 50,    3607-3613.-   U.S. Pat. No. 6,403,141. Method of obtaining long shelf life liquid    egg products. Liot, M.

1-24. (canceled)
 25. A shelf-stable liquid egg material, comprising: aliquid egg material enriched with an antibody at a hyperimmunized titer;and additional ingredients comprising: an antibody protectant; anantioxidant; and an antimicrobial agent, wherein the additionalingredients are present in concentrations effective to extend shelf lifeof the liquid egg material.
 26. The liquid egg material of claim 25wherein the liquid egg material comprises inner content of an egg thatis selected from the group consisting of whole egg, egg yolk, and eggwhite.
 27. The liquid egg material of claim 25 wherein the antibodyprotectant is selected from the group consisting of glycerol, propyleneglycol, ethylene glycol, and combinations thereof.
 28. The liquid eggmaterial of claim 25 wherein the antibody protectant comprises glycerol.29. The liquid egg material of claim 25 wherein the antimicrobial agentis selected from the group consisting of potassium sorbate,methylparaben, and propylparaben.
 30. The liquid egg material of claim25 wherein the antimicrobial agent comprises potassium sorbate.
 31. Theliquid egg material of claim 25 wherein the antimicrobial agentcomprises propylparaben.
 32. The liquid egg material of claim 25 whereinthe antimicrobial agent comprises methylparaben.
 33. The liquid eggmaterial of claim 25 wherein the antimicrobial agent comprises anessential oil.
 34. The liquid egg material of claim 25 wherein theantioxidant is selected from the group consisting of tocopherol, gallicacid ester, dibutyl hydroxyl toluene (BHT), butyl hydroxy anisol (BHA),ascorbic acid (vitamin C), vitamin E, and combinations thereof.
 35. Theliquid egg material of claim 25 wherein the antibody protectant ispresent in the liquid egg material at a final concentration of fromabout 1% to about 60% v/v, the antioxidant is present in the liquid eggmaterial at a final concentration of from about 0.01% w/v to about 5%w/v, and the antimicrobial agent is present in the liquid egg materialat a final concentration of from about 0.01% to about 60% w/v.
 36. Theliquid egg material of claim 25 further comprising a digestive enzyme.37. The liquid egg material of claim 25 further comprising a digestiveenzyme selected from the group consisting of soybean trypsin inhibitor,a chymotrypsin inhibitor, a carboxypeptidase inhibitor, a papaininhibitor, and a pepsin inhibitor.
 38. The liquid egg material of claim25 further comprising an alkalinizing agent.
 39. The liquid egg materialof claim 25 further comprising an alkalinizing agent selected from thegroup consisting of sodium carbonate, sodium bicarbonate, calciumcarbonate, magnesium carbonate, and combinations thereof.
 40. The liquidegg material of claim 25 wherein: the antimicrobial agent comprisessodium chloride, potassium sorbate, propylparaben, and methylparaben;the antibody protectant comprises glycerol; and the antioxidantcomprises vitamin E and vitamin C.
 41. The liquid egg material of claim25 wherein: the antimicrobial agent comprises about 10% w/v sodiumchloride, about 0.1% w/v potassium sorbate, about 0.1% w/vpropylparaben, and about 0.1% w/v methylparaben; the antibody protectantcomprises about 20% v/v glycerol; and the antioxidant comprises about0.1% w/v vitamin E and about 0.1% w/v vitamin C.
 42. The liquid eggmaterial of claim 25 wherein the antibody comprises an IgY antibody. 43.A method of making the shelf-stable liquid egg material as described inclaim 25, comprising: inducing an immune response in an egg-producinganimal with at least one antigen to produce an antibody at ahyperimmunized titer; collecting an egg from the egg-producing animalfollowing induction of the immune response; processing inner content ofthe egg to obtain a liquid egg material; and adding to the liquid eggmaterial in concentrations effective to extend shelf life of the liquidegg material: an antibody protectant; an antioxidant; and anantimicrobial agent.
 44. A method of using a shelf-stable liquid eggmaterial as described in claim 25, comprising administering the liquidegg material to an animal to provide passive immunity to the animal.